Ionizing radiation induces a motile phenotype in human carcinoma cells in vitro through hyperactivation of the TGF-beta signaling pathway

Radiotherapy, a major treatment modality against cancer, can lead to secondary malignancies but it is uncertain as to whether tumor cells that survive ionizing radiation (IR) treatment undergo epithelial-mesenchymal transition (EMT) and eventually become invasive or metastatic. Here, we have tested the hypothesis that the application of IR (10 MeV photon beams, 2-20 Gy) to lung and pancreatic carcinoma cells induces a migratory/invasive phenotype in these cells by hyperactivation of TGF-beta and/or activin signaling. In accordance with this assumption, IR induced gene expression patterns and migratory responses consistent with an EMT phenotype. Moreover, in A549 cells, IR triggered the synthesis and secretion of both TGF-beta 1 and activin A as well as activation of intracellular TGF-beta/activin signaling as evidenced by Smad phosphorylation and transcriptional activation of a TGF-beta-responsive reporter gene. These responses were sensitive to SB431542, an inhibitor of type I receptors for TGF-beta and activin. Likewise, specific antibody-mediated neutralization of soluble TGF-beta, or dominant-negative inhibition of the TGF-beta receptors, but not the activin type I receptor, alleviated IR-induced cell migration. Moreover, the TGF-beta-specific approaches also blocked IR-dependent TGF-beta 1 secretion, Smad phosphorylation, and reporter gene activity, collectively indicating that autocrine production of TGF-beta(s) and subsequent activation of TGF-beta rather than activin signaling drives these changes. IR strongly sensitized cells to further increase their migration in response to recombinant TGF-beta 1 and this was accompanied by upregulation of TGF-beta receptor expression. Our data raise the possibility that hyperactivation of TGF-beta signaling during radiotherapy contributes to EMT-associated changes like metastasis, cancer stem cell formation and chemoresistance of tumor cells.